Dynamic Measurement of Protein Translation in Mycobacteria Using Nontargeted Stable Isotope Labeling in Combination with MALDI-TOF Mass Spectrometry-Based Readout

Understanding the metabolic pathways of mycobacteria is essential to identify novel antibiotics and to compose synergistic antibiotic regimens against tuberculosis, one of the world’s most deadly infectious diseases with >1.7 Mio yearly deaths. We present a novel proteomics approach for the dynamic measurement of the nascent fractions of specific proteins. We use nontargeted stable isotope incorporation to label the nascent proteins after adding glycerol-1,3-¹³C₂. The analysis is performed using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with a self-programmed script, allowing quantitative data. We compared the de novo synthesis of proteins under regular growth conditions and the effect of four antimicrobials, including rifampicin as a first-line drug, linezolid and bedaquiline as second-line drugs, and benzithiazinone-043 as promising drug candidates against tuberculosis. Changes in the synthesis of individual proteins, either due to antimicrobial action or due to regulations in the organism, can be followed in high temporal resolution within the 1/2 doubling cycle of mycobacteria. The analysis of de novo protein synthesis offers a fast screening and testing tool, allowing assessment of the onset and extent of antimycobacterial activity or regulatory phenotypes in different organisms. Due to the untargeted approach, it can be used in model strains and clinical isolates alike and does not require genetic modifications. The dynamic readout and labeling reveal the onset of action of drugs or drug candidates and allow for the prediction of synergistic effects of several substances.